Toll-like receptor-4/p38 MAPK signaling in the dorsal horn contributes to P2X4 receptor activation and BDNF over-secretion in cancer induced bone pain.

Our previous research suggested that the P2X4 receptor (P2X4R) expression in microglia was involved in the activation of toll-like receptor-4 (TLR4) in the dorsal horn in the rat model of cancer induced bone pain (CIBP). In this study, we focused on whether TLR4- mitogen-activated protein kinases, p38 (p38 MAPK) contributes to P2X4R activation and brain-derived neurotrophic factor (BDNF) over-secretion in CIBP. In in vitro experiment, the results showed that BDNF expression evoked by ATP stimulation was dependent on TLR4-p38. In in vivo experiment, the results demonstrated that an intrathecal injection of TLR4 siRNA alleviated nociception induced by lipopolysaccharide (LPS) plus ATP or CIBP with decreased expression of P2X4R, TLR4, BDNF, interleukin-6 (IL-6) and phosphorylated-p38 MAPK (p-p38 MAPK). Moreover, injection with p38MAPK inhibitor SB203580 resulted in an identical pattern compared with treatment with TLR4 siRNA. Our results demonstrate that the activation of TLR4-p38MAPK-P2X4R signaling in microglial possibility plays an important role in the process of nociceptive transmission in CIBP, suggesting new mechanism and potential therapeutic targets for CIBP.

MCP-1 stimulates spinal microglia via PI3K/Akt pathway in bone cancer pain.

Accumulating evidence suggests that chemokine monocyte chemoattractant protein-1 (MCP-1) is significantly involved in the activation of spinal microglia associated with pathological pain, at the same time that the phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/Akt) pathway localized in spinal microglia is involved in both neuropathic and inflammatory pain. However, whether there is a connection between MCP-1 and the PI3K/Akt pathway and in their underlying mechanisms in bone cancer pain (BCP) has not yet been elucidated. In the current study, we investigated the expression changes of p-Akt in microglia and OX-42 (microglia marker) after being stimulated with MCP-1 in vitro, as well as in a BCP model that was established by an intramedullary injection of mammary gland carcinoma cells(Walker 256 cells) into the tibia of rats. We observed a significant increase in expression levels of p-Akt and OX-42 in microglia as well as in spinal dorsal horns of BCP rats. Furthermore, the intrathecal administration of an anti-MCP-1 neutralizing antibody or PI3K inhibitor LY294002 reduced the expression of p-Akt or OX-42, and LY294002 attenuated the mechanical allodynia of BCP rats. These results suggest that MCP-1 may stimulate spinal microglia via the PI3K/Akt pathway in BCP.

P2X4 receptor in the dorsal horn partially contributes to brain-derived neurotrophic factor oversecretion and toll-like receptor-4 receptor activation associated with bone cancer pain.

Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor-α (TNFα) following activation of toll-like receptor-4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain-derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real-time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX-42, phosphorylated-p38 MAPK (p-p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p-p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP.

Activation of spinal TDAG8 and its downstream PKA signaling pathway contribute to bone cancer pain in rats.

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Brain-derived neurotrophic factor modulates N-methyl-D-aspartate receptor activation in a rat model of cancer-induced bone pain.

Brain-derived neurotrophic factor (BDNF) released within the spinal cord induces phosphorylation of N-methyl-D-aspartate (NMDA) receptors on the spinal cord neurons. This process is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about the role of BDNF and NMDA receptors in cancer-induced bone pain (CIBP), whose features are unique. This study demonstrates a critical role of the BDNF-modulated NMDA subunit 1 (NR1) in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP, both in the spinal cord and in the dorsal root ganglia (DRG). We selectively suppressed BDNF expression by RNA interference (RNAi) using intrathecal administration of BDNF small interfering RNA (siRNA). Then, we assessed mechanical threshold and spontaneous pain in CIBP rats. Real-time PCR, Western blotting, and fluorescent immunohistochemical staining were used to detect BDNF or NR1 both in vivo and in vitro. BDNF and phospho-NR1 were expressed under CIBP experimental conditions, with expression levels peaking at day 6 (BDNF) or 9 (NR1). Intrathecal BDNF siRNA prevented CIBP at an early stage of tumor growth (days 4-6). However, at later stages (days 10-12), intrathecal BDNF siRNA only attenuated, but did not completely block, the established CIBP. BDNF-induced NMDA receptor activation in the spinal cord or DRG leads to central sensitization and behavioral hypersensitivity. Thus, BDNF might provide a targeting opportunity for alleviating CIBP.

Minocycline-induced reduction of brain-derived neurotrophic factor expression in relation to cancer-induced bone pain in rats.

Previous studies have suggested that the release of brain-derived neurotrophic factor (BDNF) from microglia in spinal cord is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. This study demonstrates a critical role of minocycline (a potent inhibitor of microglial activation)-modulated BDNF in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, minocycline was administered intrathecally from day 4 to day 6 (early stage) or from day 10 to day 12 (later stage), after carcinoma cell inoculation. Real-time PCR, Western blots, and double immunofluorescence were used to detect the expression of OX-42 (marker of activated microglia), phosphorylated p38-MAPK (p-p38), and BDNF. We found that intrathecal minocycline could prevent CIBP at an early stage of tumor growth (from day 4 to day 6). However, at the late stage (from day 10 to day 12), intrathecal minocycline had no effect. Moreover, the expression of OX-42 and BDNF under CIBP, peaking on day 6, were all reduced after minocycline injection from day 4 to day 6. The ability of minocycline-induced reduction of BDNF in the induction of behavioral hypersensitivity could provide an opportunity for alleviating CIBP.

[The role of brain-derived neurotrophic factor in pain facilitation and spinal mechanism in rat model of bone cancer pain].

OBJECTIVE: To investigate the role of brain-derived neurotrophic factor (BDNF) in pain facilitation and spinal mechanisms in the rat model of bone cancer pain. METHODS: The bone cancer pain model was developed by inoculated Walker 256 mammary gland carcinoma cells into the tibia medullary cavity. Sixty SD female rats were divided into 5 groups (n = 12 each) randomly; group I: control group (sham operation); group II: model group; group III: control group + anti-BDNF intrathecal (i.t.); group IV: model group + control IgG i.t.; group V: model group + anti-BDNF i.t.. Anti-BDNF or control IgG was injected i.t. during 7 to 9th day. Von-Frey threshold was measured one day before operation and every 2 days after operation. On the 9th day after threshold tested, rats were sacrificed after i.t. injection of either anti-BDNF or control IgG, the lumbar 4-6 spinal cord was removed. The expression of the spinal BDNF and the phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) were detected by immunohistochemistry assay and Western-Blot. Co-expression pattern of BDNF and p-ERK1/2 were determined by double-labeling immunofluorescence. RESULTS: We demonstrated the coexistence of BDNF and p-ERK1/2 in the spinal cord of rats. From the 7 to 9th day after operation, von-Frey threshold in groups II and IV was significantly lower than that in group I and group V (P < 0.01), group V was remarkly higher than that in group IV (P < 0.01). The spinal BDNF and p-ERK1/2 expression in group II or IV were significantly increased compared with that in group I or V (P < 0.01), intrathecal anti-BDNF was significantly suppressed BDNF and p-ERK1/2 expression (P < 0.01). CONCLUSION: BDNF and p-ERK1/2 was coexistence in the spinal cord of rats, and it maybe involved in the bone cancer pain.

Cancer-induced bone pain sequentially activates the ERK/MAPK pathway in different cell types in the rat spinal cord.

BACKGROUND: Previous studies have demonstrates that, after nerve injury, extracellular signal-regulated protein kinase (ERK) activation in the spinal cord-initially in neurons, then microglia, and finally astrocytes. In addition, phosphorylation of ERK (p-ERK) contributes to nociceptive responses following inflammation and/or nerve injury. However, the role of spinal cells and the ERK/MAPK pathway in cancer-induced bone pain (CIBP) remains poorly understood. The present study analyzed activation of spinal cells and the ERK/MAPK pathway in a rat model of bone cancer pain. RESULTS: A Sprague Dawley rat model of bone cancer pain was established and the model was evaluated by a series of tests. Moreover, fluorocitrate (reversible glial metabolic inhibitor) and U0126 (a MEK inhibitor) was administered intrathecally. Western blots and double immunofluorescence were used to detect the expression and location of phosphorylation of ERK (p-ERK). Our studies on pain behavior show that the time between day 6 and day 18 is a reasonable period ("time window" as the remaining stages) to investigate bone cancer pain mechanisms and to research analgesic drugs. Double-labeling immunofluorescence revealed that p-ERK was sequentially expressed in neurons, microglia, and astrocytes in the L4-5 superficial spinal cord following inoculation of Walker 256 cells. Phosphorylation of ERK (p-ERK) and the transcription factor cAMP response element-binding protein (p-CREB) increased in the spinal cord of CIBP rats, which was attenuated by intrathecal injection of fluorocitrate or U0126. CONCLUSIONS: The ERK inhibitors could have a useful role in CIBP management, because the same target is expressed in various cells at different times.